The present invention relates to assays that employ an enzyme label or tag that acts on a substrate by obtaining electrons from an electrode (electrocatalysis) and an apparatus for use in such assays.
Immunoassay techniques are based on the ability of antibodies to form complexes with the corresponding antigens or haptens. This property of highly specific molecular recognition of antigens by antibodies leads to high selectivity of assays based on immune principles. The high affinity of antigen-antibody interactions results in great sensitivity of immunoassay methods. The use of a label or indicator to verify that an antigen/antibody interaction has occurred is the basis for immunoassay methods.
Immunoassay techniques have been used mainly in clinical analyses and medical diagnostics. However, immunoassay applications in other areas such as environmental control, food quality control, etc. are expanding. Certain limitations in assaying techniques due to existing procedures have limited somewhat the expansion into such other areas.
In this respect, during the last few years a significant number of publications have dealt with non-conventional (alternative) immunoassay techniques designed to expand the accuracy or applicability of immunoassays. In most cases the development of alternative immunoassay techniques aims at improvements in performance of conventional immunoanalysis. Often such improvement attempts are directed to decreasing analysis times, increasing assay sensitivity, and simplifying and automating assay procedures.
For example, the utilization of enzymes able to catalyze electrochemical reactions by direct (mediatorless) mechanism (bioelectrocatalysis) would allow for the detection of immuno-interactions in real time. Such applications of bioelectrocatalysis in the development of immunosensors are based on the self-assembling or displacement of molecule/label complexes or xe2x80x9cmolecular transducersxe2x80x9d on the surface of an electrode that has been modified by immunospecies that bind the complex. Ordinarily these immunospecies would be complimentary to the immunoconjugate which includes the electrocatalytically active enzyme-label.
Antigen immobilized on the electrode surface interacts with the enzyme-labeled antibody which results in the attachment of the enzyme to the electrode surface. Attachment of the electrocatalytic active enzyme on the electrode surface initiates, in the presence of a substrate, an electrocatalytic reaction. Therefore, the formation of an antigen-labeled antibody complex on the electrode surface is accompanied by an assembling of the molecular transducing layer. The rate of electron transfer can be limited by the efficiency of electrical connection between the enzyme-label and the electrode surface, which is already modified by the immobilized immunospecies.
A potentiometric immunosensor based on mediatorless bioelectrocatalysis has been utilized which employed laccase enzyme as an electrocatalyst-label. The electrocatalytic property of the enzyme in the reaction of oxygen electroreduction (reaction 1) allowed the detection of the biospecific interaction of a laccase-labeled receptor, or antibody, with a ligand modified electrode. Formation of a complex between the laccase labeled antibody and antigen on the electrode surface results in a considerable shift in electrode potential due to the catalytic reduction of over voltage. Analysis was performed in a competitive scheme, and a single measurement was made with 20 minutes. Such a potentiometric immunoassay does not require an electrochemically active mediator. The reaction substrates were atmospheric oxygen and electrons that were transferred directly from the electrode to the oxygen molecule via the active site of the enzyme. Insulin was used as a model analyte.
In the above immunoassay sensor, the electron which is the xe2x80x9csecond substratexe2x80x9d of enzymatic reaction can be captured by the enzyme-label only from the electrode surface. Therefore, only molecules intimately attached to the electrode surface generate electrochemical signal. The rate of attachment of electrocatalyst molecules to the electrode surface is proportional to the rate of formation of the immuno-complex on the electrode surface. The rate of attachment of electrocatalyst molecules to the electrode surface is proportional to the rate of formation of the immuno-complex on the electrode surface. The rate of immunointeraction on the electrode surface can be directly monitored by amperometric or potentiometric mode.
However, assays based on mediatorless bioelectrocatalysis are limited in that primarily one of the two assay procedures set forth below are utilized and only a single assay measurement may be taken before the electrode is regenerated or replaced by a new electrode. The two competitive assay procedures are:
(a) Competitive Immunoassay With an Initial Label-free Electrodexe2x80x94An electrode having no attached analyte/enzyme label is utilized as a starting point and a measured amount of analyte media along with a measured quantity of analyte/enzyme label are assayed by a competitive binding assay procedure. After maximum association with the electrode has occurred the amperometric or potentiometric measure result is compared to that of an electrode having 100% analyte/enzyme label associated. The difference in measurements corresponds proportionally to the amount of analyte in the media being assayed. and
(b) Displacement Immunoassay With an Initial Label-loaded Electrodexe2x80x94An electrode having the maximum amount of attached analyte/enzyme label (a filly loaded electrode) is utilized as a starting point and a measured amount of analyte media is assayed by a competitive binding assay procedure. After maximum displacement of the analyte/enzyme label from the electrode by the analyte of the media has occurred the amperometric or potentiometric measure result is compared to that of the initial fully loaded electrode (having 100% analyte/enzyme label associated). The difference in measurements corresponds proportionally to the amount of analyte in the media being assayed.
In addition to the laccase enzyme label, the potentiometric immunosensor employing peroxidase as an electrocatalyst-label has also been developed. The basic principle is the same as for the laccase based immunosensor. The electrode surface is modified by an immobilized antigen (rabbit IgG). The peroxidase-antibody conjugate associates with the antigen on the electrode surface. Once added to the media, and on reaching the electrode surface, the antibody-conjugated peroxidase starts to catalyze the electro-reduction of hydrogen peroxide. This results in an increase (anodic shift) in the electrode potential.
Both the laccase and peroxidase label immunosensors based on bioelectrocatalytic detection (as discussed above) allow direct detection of immunointeraction in real time. However, these sensors must be regenerated or replaced (e.g., disposable sensors) after each measurement. Accordingly, such immunoassay procedures do not allow continuous monitoring of the analyte. In addition, such procedures are a multi-stage process that result in a general complexity of analysis and require a highly qualified technician to conduct the assay.
Accordingly, there is a need for immunoassay procedures that can be continuous, particularly automatable procedures or procedures that do not require highly qualified technicians to conduct the assay.
An object of the present invention is to provide an improved bioelectrocatalysis immunoassay apparatus for detecting an analyte, the apparatus comprising a sensing device with an electrode, wherein the sensing device has the ability to monitor changes in the amount of analyte in a liquid without requiring regeneration or replacement of the electrode or of the reagents used in the assay. Preferably, the sensing device of the apparatus is capable of multiple intermittent and/or continuous immunoassay measurements of the same analyte without a requirement for regeneration or replacement of the electrode or other reagents.
In one aspect the apparatus is for detecting an analyte that is an antigen, antibody or hapten by use of a labeled detection compound that is labelled with an electrocatalytic enzyme, wherein the labeled detection compound is either the analyte or the binder for the analyte, and wherein the apparatus provides an electrode to which is permanently affixed (i) a binder for the analyte in the case wherein the labeled detection compound is a labeled analyte or (ii) the analyte in the case wherein the labeled detection compound is a labeled binder for the analyte. If such detection compound becomes closely associated with the electrode of the apparatus, the electrocatalytic enzyme label or tag will interact with the electrode to cause a detectable electrical change for the electrode. In one embodiment the analyte is the same antigen, antibody or hapten as the detection compound (wherein the detection compound is labeled with an electrocatalytic enzyme or tag) and some of the fixed amount of the labelled detection compound complex that is detectable by the electrode due to bioelectrocatalysis competes with the analyte for binding to the binder of the electrode. Alternatively, the binder on the electrode and the analyte are the same antigen, antibody or hapten and compete for binding to the detection compound that is labelled with the electrocatalytic enzyme.
In accordance with the above apparatus (depending upon whether the analyte is the same as the binder or the same as the detection compound) the analyte will bind to either the binder or to the detection compound. In either event, the amount of the detection compound that attaches to the binder is inversely proportional to the concentration of the analyte in the sample being analyzed. The amount of the detection compound which is bound to the binder of the electrode is indirectly measurable by bioelectrocatalysis and the amount of analyte which is present in the sample is therefore detectable by the amount of bioelectrocatalysis occurring at the electrode""s surface. In each of the above cases (i.e., (i) when the binder and analyte are the same and (2) when the binder and the detection compound are the same, the electrode only transfers an electron to the label as a substrate when the labeled detection compound is attached to the binder affixed to the electrode. Therefore, a shift in potential or current, can be observed by the amount of labeled detection compound that binds to the binder on the electrode, which amount of labeled detection compound bound to the binder on the electrode is inversely proportional to the amount of analyte in the sample. When current is measured instead of potential at the electrode, the current is proportional to the amount of laccase label attached to the electrode. Measurement of potential is preferred since amperometric measurement of the electrode signal takes into consideration the surface area of the electrode and the density of the laccase label attached to the electrode. Thus, potential measures are usually more accurate, but sometimes amperometric measures are more accurate and should be used. The ordinary practicioner in this field would know when to use a particular electrode type. Thus, when the terms xe2x80x9cpotentiometric electrodexe2x80x9d and xe2x80x9cpotentiometric assayxe2x80x9d and the like are used in this application and claims the word xe2x80x9camperometricxe2x80x9d may be substituted for xe2x80x9cpotentiometricxe2x80x9d.
A further object of the invention is to provide an improved bioelectrocatalysis immunoassay sensing device wherein the electrode is encased by a housing member comprising at least one porous or semi-porous surface, such as a semipermeable membrane, that is permeable for an analyte and impermeable to the labelled detection compound, preferably a detection compound that is labelled with an electrocatalytic enzyme. In a preferred aspect the sensing device comprises a particular quantity of the labelled detection compound which is enclosed within the housing member, which labelled detection compound may contact the electrode when the sensing device is placed in a liquid or gaseous medium and the at least one porous or semi-porous surface is impermeable to the labelled detection compound.
A preferred object of the invention is to provide an apparatus for immuno-determination of target analyte (antigen, antibody or hapten) in an analyte sample, where said apparatus comprises
(a) a sensing device comprising:
(i) a potentiometric working electrode at least one surface of which is located within said housing member, wherein said electrode is connected to a potentiometric measuring circuit and said electrode has the ability to provide an electron to an enzyme label which will deliver the electron to a first substrate for the enzyme label, and said electrode has permanently affixed to at least one of its surfaces a binder for at least a detection compound which is labelled with an electrocatalytic enzyme;
(ii) a housing member comprising at least one surface that is permeable to an analyte and impermeable to the labelled detection compound, wherein the detection compound is a member selected from the group consisting of (a) a binder for the analyte (in the case where the binder on the electrode is the analyte) and (b) the analyte (in the case where the binder on the electrode is a binder for the analyte), and in each case the detection compound is labelled with an electrolytic enzyme or tag; and
(iii) an internal media which is located within the housing member and which is a gel or liquid containing a pre-determined amount of labelled detection compound as herein above described, or capable of containing a pre-determined amount of the labelled detection compound; and
(b) an electrochemical reference electrode connected to a potentiometric measuring circuit, and wherein the internal media of (iii) or the analyte sample comprises a second substrate for the enzyme label or tag, such as oxygen. The analyte will either bind to the binder or to the detection compound.
In a preferred aspect, the apparatus further comprises at least one measuring device which is connected directly or indirectly to the sensing device and/or the reference electrode. Preferably, the at least one measuring device is a member selected from a digital voltmeter or other similar a measuring device. In one aspect said measuring device is interfaced with a personal computer as well as the sensing element and the electrochemical reference probe. In a preferred aspect, the measuring device is also connected to a member selected from (i) a signal recorder which is an X-T recorder, (ii) a microprocessor based data acquisition system with a digital display, and (iii) a personal computer.
Additionally, an object of this invention is provide a sensing device comprising an external housing with at least one semipermeable surface and having within the housing device at least one surface of a working electrode comprised of an electrode body made by electrochemically inert electro-conductive material modified by a binder immobilized on its surface which will bind to at least a detection compound which is labelled with an electrocatalytic enzyme, and the binder may be the same as the analyte or may be a binder for the analyte. In a preferred aspect, the binder is a binder for both the analyte and the labeled detection compound, whereby the binder will reversibly bind individually to the analyte or to the labelled detection compound which will each compete to be bound by the binder. Preferably, the detection compound is labelled with the laccase enzyme which can use oxygen and electrons from the electrode as substrates.
Another object of the invention is a sensing element which comprises (a) a potentiometric working electrode, (b) an external housing with at least one surface that is a semipermeable surface (such as a diffusion membrane) through which an analyte may diffuse and is impermeable to at least one detection compound which is labelled with an electrolytic enzyme or tag and the detection compound will bind to either the binder of the electrode, or to both the binder on the electrode and the analyte of the sample, and (c) an electrochemical reference electrode. In a preferred aspect the sensing element further comprises an internal media, which is a gel or liquid, and a fixed quantity of the labelled detection compound, or comprises a means for inserting a quantity of labelled detection compound and/or the internal media comprising a fixed quantity of the labelled detection compound.
Another object of the invention is to provide a portable biosensor capable of continuously detecting a target analyte in a wide range which operates in a continual potentiometric mode from which multiple potentiometric measurements are available that correspond to the amount of analyte present in a given sample being assayed.
In yet another aspect, an object of the present invention is to provide a method for intermittently or continuously conducting immunoassay measurements wherein a plurality of different measurements for an analyte are available for a single electrode without requiring regeneration of said electrode and other reagents. In particular, such object is accomplished by a method of determination a target analyte based on displacement activity of the target analyte and a potentiometric mode comprising the following steps:
(a) immersing of the intermediate and/or continuous bioelectrocatalysis immunoassay sensing element (described above) in a assay medium containing the target analyte,
(b) allowing the target analyte of the assay medium to diffuse through the diffusion membrane of the sensing device and travel to the surface of the working electrode,
(c) permitting an immuno-equilibrium to be established within the sensing device with respect to the amount of target analyte present in the assay medium due to displacement of some or all of a labelled detection compound from the binder on the surface of the electrode by the target analyte which target analyte becomes bound to the binder on the surface of the sensing element in the case where the detection compound is the same as the analyte or the target analyte becomes bound to the detection compound in the case where the detection compound is a binder for the analyte,
(d) measuring at least one shift of the electrode potential caused by the displacement of some or all of the labeled detection compound from the electrode""s surface and the resulting diminishment or absence of electrocatalytic properties caused by the label of detection compound,
(e) determining the potentiometric sensor response which is proportional to the degree of displacement of the labeled detection compound from the binder on the surface of the working electrode caused by competitive binding of the target analyte, and
(f) determining the concentration of the target analyte in the external media from the potentiometric sensor response as compared with the control electrochemical reference electrode.
In yet further aspect, an object of the present invention is to provide a method for intermittently or continuously conducting competitive immunoassay measurements wherein a plurality of different measurements are available for a single electrode without requiring regeneration of said electrode. In the situation where the amount of target analyte changes to become more or less concentrated in a continuous monitoring process, a competitive binding immunoassay can be utilized. In particular, such object is accomplished by a method of determination the variations in amount of a target analyte based on displacement activity of the enzyme-labeled detection compound (i.e., labeled analyte) or from a binder for the analyte and the detection compound which binder is on the working electrode and corresponding potentiometric responses to such attachment changes, which method comprises the following steps:
(a) immersing of the intermediate and/or continuous bioelectrocatalysis immunoassay sensing element (described above) in a assay medium which may vary continuously with respect to the concentration of the target analyte present,
(b) allowing the target analyte of the assay medium to diffuse through the diffusion membrane of the sensing device and travel to the surface of the working electrode,
(c) permitting an immuno-equilibrium to be established within the sensing device with respect to the amount of target analyte present in the assay medium due to (i) displacement of some or all of a labeled detection compound (e.g., labeled analyte) that is reversibly bound to a binder immobilized on the surface of the electrode by the target analyte which target analyte becomes reversibly bound to the binder immobilized on the surface of the sensing element upon displacing the labeled detection compound, or (ii) displacement of some of the reversibly bound target analyte from the binder on the surface of the electrode by some of the fixed amount of the labeled detection compound that is present within the sensing device due to a lowering of the concentration of the target analyte which causes a shift in its binding equilibrium and degree of binding with the binder on the surface of the electrode,
(d) measuring at least one shift of the electrode potential caused by the displacement occurring on the surface of the working electrode of either the reversibly bound labeled detection compound or the reversibly bound target analyte and resulting changes in electrocatalytic properties occurring at the surface of the electrode,
(e) determining the potentiometric sensor response which is proportional to said binding displacement on the surface of the working electrode,
(f) determining changes in the concentration of the target analyte in the external media from changes in the potentiometric sensor response as compared with the control electrochemical reference electrode and prior measurements from the working electrode.
In accordance with one aspect of the invention, there is provided an assay procedure wherein a sample containing analyte (a substance to be determined) placed in contact with an electrode which supports a binder for the analyte, which binder has bound thereto a labeled form of the analyte in which the label is an electrocatalytic enzyme (an enzyme that acts on a substrate by obtaining electrons from the electrode) and wherein such electrode is also contacted with a substrate for such enzyme label. The analyte in the sample displaces some or all of the reversibly bound labeled analyte from the binder, with the amount of binding displacement being directly related to the amount of analyte in the sample. Displacement of labeled analyte results in a change in voltage between such electrode and a reference electrode and such change in voltage is a measure of the amount of analyte in the sample.
In a preferred embodiment, the electrode having supported thereon the binder having bound thereto a labeled analyte is present in a chamber which includes a member or wall which is permeable to analyte but which is impermeable to the enzyme labeled analyte.
The sensing element is comprised of an electrode made by electrochemically inert electroconductive material (such as different carbon based materials, gold, different electroconductive polymers) modified by immobilized binder (such as the analyte) that is directly attached to the surface of the electrode and may be the same as the analyte or as a detection compound.